Alphaviruses include important and emerging human pathogens such as Chikungunya virus, Mayaro virus and the encephalitic alphaviruses. There are currently no licensed antiviral therapies or vaccines, and new approaches and information are needed to develop antiviral strategies. Alphaviruses are small enveloped plus-sense RNA viruses that infect cells via clathrin-mediated uptake and low pH-triggered fusion, and replicate in the cytoplasm. The capsid protein selectively packages the RNA genome to form the highly organized nucleocapsid. Budding through the plasma membrane envelopes this core in a membrane containing an organized lattice of the E2/E1 envelope proteins. The resulting virus particle has high specific infectivity, attesting to the specificity and high-quality control of assembly. Gaps in our knowledge include the mechanisms that promote and constrain RNA packaging and nucleocapsid formation, which occur in the presence of abundant cellular and subgenomic RNAs and high-levels of cytoplasmic capsid protein. While it is clear that alphavirus entry involves receptor-mediated endocytosis, there is little molecular information on virus-receptor interactions. This grant will address these critical steps in 3 inter-related aims: 1. Mechanism by which berberine inhibits alphavirus nucleocapsid formation. Our preliminary data identify a novel and selective effect of the small molecule berberine on alphavirus nucleocapsid formation. We will define berberine?s mechanism and use this inhibitor to analyze the biology of nucleocapsid assembly. 2. RNA-capsid protein interactions that drive selective viral RNA packaging in virus-infected cells. We have used 4-thiouridine labeling and photoactivation to specifically crosslink viral RNA with capsid protein in the cytoplasmic, cellular nucleocapsid and viral nucleocapsid pools. Our preliminary results indicate that we have mapped capsid binding sites on the viral RNA with nucleotide precision, and identified both the known packaging sequence and a set of novel sites in the genomic RNA. We will analyze these sites for sequence/structural motifs and compare across alphavirus clades. We will test the roles of these novel sites in mediating specific viral RNA packaging, nucleocapsid assembly, and packaging of reporter RNAs. 3. Molecular features of NRAMP2 as an alphavirus receptor. The multi-pass iron transporter NRAMP2 has been identified as the receptor for Sindbis virus (SINV), but the structure and function of the receptor vs. virus infection are not understood. We will determine the properties of the receptor required to promote endocytic uptake and infection. We will take advantage of recent NRAMP structural studies to test the role of iron binding, pH regulation, and transporter conformational changes in virus receptor activity, and map the SINV binding sites on the extracellular loop(s) of NRAMP2. The results of these aims will provide novel and fundamental information on the alphavirus lifecycle and the host factors that promote it.